extraction and characterization of galactomannan from guar

TEXAS TECH UNIVERSITY

Extraction and Characterization of

Galactomannan from Guar Seeds

Noureddine Abidi, PhD.

[email protected]

Managing Director and Professor

Fiber and Biopolymer Research Institute

Dept. of Plant and Soil Science

Texas Tech University

Outline

❑ Introduction

❑ Objectives

❑ Extraction of Guar Gum

❑ Characterization of Guar Gum

❑ Conclusions

Introduction

❑ Guar gum is a natural water dispersible hydrocolloid that has great

thickening power when dispersed in water

❑ It is extracted from the seeds of Cluster Bean/Guar

(Cyamopsis tetragonoloba (L))

❑ Growing regions: India

Pakistan

USA - Texas and Oklahoma

Introduction

❑ Hydraulic fracturing in oil well drilling:

- Mixed with the fracturing fluid to harvest shale gas and oil

❑ Food industry

- Stabilizer in frozen (ice cream) and baked foods

- Thickener for salad dressing due to high viscosity, acid stability & cold water

dispersibility

❑ Pharmaceutical, textile, and paper industries

Major Commercial Uses of Guar Gum

Picture source: http://2.imimg.com/data2/QR/DQ/MY-1031926/we-offer-250x250.jpg, http://www.therecklessoptimist.com/wp-content/uploads/2010/10/Milkshake-12447500.jpg, http://momentumbooks.com.au/wp-

content/uploads/2013/10/squash-and-apple-soup.jpg, http://images2.alphacoders.com/130/130230.jpg, http://www.pumpkinseedbulkfood.com/upload/images/Troyer_Pictures/donna_sharp_images/BREAD_PHOTO.jpg

Introduction

❑ Hydraulic fracturing uses high pressure fluid to

crack open hydrocarbon bearing zones in shale

rock formations

❑ Guar gum is one of the most popular polymer used

in aqueous based fracturing fluids since it has a

great viscosifying property

❑ Guar gum thicken the fracturing fluid to retain the

graded sand/ proppant in suspension and prevent

the settling of the proppant

❑ The proppant helps to keep the fracture open,

creating a permeable route for the oil or gas to flow

to the well bore

Hydraulic Fracturing

http://www.halliburton.com/public/pe/con

tents/Brochures/Web/H09347.pdf

Picture source:

https://student.societyforscience.org/article/

fracking-fuels-energy-debate

Introduction

❑ Guar gum increases the efficiency of the process by reducing

the friction of the system

❑ ~ 9000 kg of guar gum is required per oil well

❑ Hydroxypropyl guar (HPG) and carboxymethyl-

hydroxypropyl guar (CMHPG) are also used in hydraulic

fracturing

❑ The organometallic cross-linked guar, HPG and CMHPG

are used when there is a need for extended fracture length❑

❑ Borate cross-linked guar and HPG provide good proppant

transportation and high temperature stability

Hydraulic Fracturing

Introduction

❑ A leguminous plant that grows up to 3 – 6 feet

❑ Produces many 5 – 12.5 cm long bean-like pods in clusters with

6 – 9 small seeds per pod

❑ Extremely drought tolerant annual crop that can be cultivated

with very limited supply of resources

Guar Plant

Picture Source: Dr. D. L. Auld – Quaker Farm, Texas Tech University

Guar Plants

Introduction

Picture source: Whistler, R.L., Hymowitz, T., (1979), Guar: Agronomy, Production, Industrial use and Nutrition (left)

Guar Seed Cross Section

40 - 46 % - Germ

38 - 45 % - Endosperm

14 - 16 % - Hull

Guar Seed -

Guar Seed

Seed Coat

Radicle

Endosperm

CotyledonGerm

Introduction

❑ Water soluble polysaccharide fraction consists of

D - galactose - 36.6%

D - mannose - 63.1 %Galactomannan = Pure guar gum

Compound Percentage

Nitrogen 0.67

Phosphorus 0.06

Ash 1.07

Water soluble polysaccharide 86.50

Water insoluble fraction 7.75

Alcohol soluble fraction 1.50

Composition of Guar EndospermSource: Chudzikowski, (1971).

Introduction

❑ Linear chain of D-mannose units linked together via a β(1 – 4)

acetal linkage and having approximately one D galactose unit for

every alternate mannose unit linked via an α(1-6) acetal linkage

❑ Molecular weight ~ 220 KDa

Picture source : http://www.fao.org/fileadmin/templates/agns/pdf/jecfa/cta/69/Guar_gum.pdf.

Chemical Structure of Guar Galactomannan

Introduction

❑ There is an increased interest in guar gum and its application,

particularly in the food and hydraulic fracturing industries

❑ Therefore, characterizing guar gum using different analytical tools is

very important to understanding the chemistry of guar galactomannan

❑ However, a limited number of studies have been conducted to

characterize the gum extracted from the currently available guar

germplasm

Objectives

1. Optimize the extraction method to minimize contamination of

galactomannan

2. Characterize guar gum using different analytical tools

3. Elucidate the physical and chemical differences between guar

cultivars

Morphology of Guar Seed

Scanning Electron MicroscopePictures:

Fiber and Biopolymer Research Institute - Texas Tech University

Extraction and Characterization of Galactomannan from Guar Seeds

Morphology of Guar Seed

Guar Seed (wet) Cross Section

Longitudinal Section Guar GermGuar Endosperm

Cotyledon

RadicleGerm

Endosperm

Seed Coat (Hull)

Guar Seed (dry)

Cross Sections of Guar Seeds

Seed Coat

Radicle

Endosperm

Cotyledon

Matador

Monument

Seed Coat

Radicle

Endosperm

Cotyledon

Matador Monument Matador Monument

Cross Sections of Guar Seed

x40 x400

x1000x100

C E

H

C

E H C E

C

E

C R

H H

R E E

C- Cotyledon, E- Endosperm, R- Radicle, H- Seed Coat/Hull

Extraction of Gum from Guar Seeds

1. Manual Extraction of Endosperm

2. Ethanol Precipitation of Whole Seed Powder


Extraction of Guar Gum


❑ FTIR spectra were collected from sample A and B to investigate the impact

of heat treatment on galactomannan

❑ Both spectra are identical and therefore initial heat treatment can be used

before the manual extraction to prevent germination during water soaking

and endosperm extraction

FTIR spectra of Guar endosperm powder separated by different methods

1. Manual Extraction of Guar Gum


Matador & Monument Seeds

Water soak overnight (80oC )

Manually separate endosperm

Dry at 105oC for 40 min

Grind endosperms and sieve the powder

Store the endosperm powder in refrigerator at 4oC

Heat Treatment at 100oC for 30 min

1. Manual Extraction of Guar Gum


❑ Level of contamination is high with this method

❑ 1740 and 1543 cm-1are attributed to C=O and NH2 functional groups

respectively (from ester, amino acids or proteins)

❑ Repetitive ethanol precipitation reduces the level of contamination

FTIR Spectra of Ethanol precipitated Matador and Monument whole seed powder

Matador Monument

2. Ethanol Precipitation of Whole Guar Seed Powder

Characterization of Guar Gum


Characterization of Guar Gum

FTIR Fourier Transform Infrared

Spectroscopy

TGAThermogravimetric Analysis

HPLCHigh Performance Liquid

Chromatography

1. Matador endosperm powder (MAT)

2. Monument endosperm powder (MON)

3. Guar galactomannan (GGM)

4. Food grade guar gum (FGG)

* The galactomannan samples extracted by ethanol precipitation of whole seed powder were not further

characterized in this study

FTIR Study:Purity of guar gum

Pictures:



FTIR Study

❑ FTIR spectra were collected for FGG, MAT, MON, GGM

❑ Vibrations 1740 and 1543 cm-1 are attributed to C=O and NH2

functional groups respectively (from ester, amino acids or proteins)

FTIR Spectra of Guar Gum

FTIR Study

FTIR spectra of MAT & E-MAT (left), MON & E-MON (right)

+MilliQ Water

(80oC)Filter Ethanol

(MAT, MON) (E-MAT, E-MON)

1543

1740

15431740

TGA Study:Thermal Behavior of Guar Gum

Pictures:



Thermal Behavior of Guar Gum

❑ All samples were conditioned at 21±1oC and 65±2% RH for at

least 24 hrs

❑ Temperature range: 40 to 600oC

❑ Heating rate : 10oC/ min

❑ Atmosphere : Nitrogen

❑ Weight losses of materials were recorded

❑ Number of replicates: 3

Thermal Behavior of Guar Gum

Percent weight loss between 150 - 450oC

Galactomannan degradation temperature

TGA Thermogram of GGM

ANOVA

❑ Weight loss at ~ 42oC and ~ 305oC are attributed to evaporation of water

and degradation of galactomannan respectively

Weight Loss Between 150- 450oC(Degradation of Galactomannan)

❑ The percent weight losses in the temperature range of 150- 450oC are

significantly different (α = 0.05)

❑ The percent weight loss in the second region could be an indication of the

galactomannan content (purity) of the material

(GGM > E-MON > E-MAT > FGG > MAT/MON)

Sample

ID

Mean weight

loss (%)

GGM 77.1 a

E-MON 72.6 b

E-MAT 68.9 c

FGG 66.4 d

MON 63.7 e

MAT 63.6 e

Values not followed by the same letter are significantly

different with α = 0.05

Galactomannan Degradation Temperature

❑ The degradation temperatures of the samples are significantly different (α=0.05)

❑ Impurities with a lower degradation temperature may induce galactomannan

to degrade at an early temperature

❑ Ethanol precipitation has significantly increased the thermal stability of

endosperm samples in both cultivars

❑ Removal of impurities as well as the mannose to galactose ratio of the samples

may have great impact on the improved thermal stability

Parameter Temperature (oC)

E-MAT 328.7 a

E-MON 324.9 b

GGM 303.9 c

FGG 300.9 d

MON 294.1 e

MAT 293.3 e


different with α = 0.05

HPLC Study:Mannose to Galactose Ratio (M/G) of Guar Gum

Pictures:



Chemical Structure of Guar Galactomannan

Picture source : http://www.fao.org/fileadmin/templates/agns/pdf/jecfa/cta/69/Guar_gum.pdf.

M/G Ratio of Guar Gum

❑ Calibration curves for galactose and mannose were developed using

different concentrations of both sugars

❑ Well-separated peaks with different retention times indicate:

▪ CarboPac PA10 column is suitable for identification of galactose and mannose

▪ The detector is suitable for quantification of galactose and mannose

Chromatogram of Mannose and Galactose with

different concentrations

Galactose

Mannose

0

20

40

60

80

100

120

14.0 16.0 18.0 20.0 22.0

nC

Retention time (min)

G M 0.25 mM

G M 0.50 mM

G M 0.75 mM

G M 1.00 mM

G M 1.50 mM

Calibration Curves for Mannose and

Galactose


❑ 10 mg of GGM was hydrolyzed using Trifluoroacetic Acid (TFA)

and Sulfuric Acid (H2SO4 )

A. 2N TFA at 120oC for 2 hrs: Wide overloaded peaks

M/G ratio: 2.46

B. 1N TFA at 120oC for 2 hrs: Wide overloaded peaks

M/G ratio: 1.72

C. 0.5M H2SO4 at 100oC for 3 hrs: Well-separated peaks

M/G ratio: 1.94

❑ Number of replicates: 3


❑ 10 mg of GGM, FGG, MAT, MON, E-MAT and E-MON samples

were hydrolyzed using 500 µL of 0.5M H2SO4 at 100oC for 3hrs :

3 replicates

❑ Samples were dried in SpeedVac at 40oC

❑ Samples were dissolved in 300µL of methanol and dried in

SpeedVac

To remove methanol soluble compounds

To prevent microbial degradation of sugars

❑ Samples were dissolved in 500µL of milliQ water

❑ Samples were analyzed using HPLC


❑ The M/G ratios of the materials are significantly different (α = 0.05)

❑ Varietal differences and purification method could change the M/G

ratio

❑ Ethanol purification may have removed galactose side branches which

may result in significantly high M/G ratios in E- MAT and E-MON

Parameter df F P M/G

Intercept 1 5248.7 0.000001

ID 5 11.2 0.000354

GGM 2.04 b

MON 2.10 b

MAT 2.16 b

FGG 2.44 a

E-MON 2.56 a

E-MAT 2.66 a

Error 12


different with α = 5% (according to Newman- Keuls test)

M/G

Conclusions

❑ Guar gum is extracted with a high level of purity with two

methods

1. Manual endosperm extraction followed by ethanol precipitation

2. Repetitive ethanol precipitation of aqueous solution of whole guar seed

powder

❑ FTIR can be used as a fast and non-destructive method to

investigate the purity of guar gum

❑ TGA study confirms that the thermal behavior of MAT and

MON are not significantly different

Conclusions

❑ Guar gum can be hydrolyzed using 0.5M H2SO4 at 100oC for 3

hours

❑ HPLC study confirms that the M/G ratios of MAT and MON

are not significantly different

❑ Method of purification has a significant influence on the M/G

ratio and thermal stability of guar gum;

Ethanol precipitation has significantly increased the M/G ratio and the

thermal stability of both MAT and MON

TEXAS TECH UNIVERSITY

Thank you

Contact information:

Noureddine Abidi, PhD.

[email protected]

Phone: 806-834-1221

Managing Director and Professor

Fiber and Biopolymer Research Institute

Dept. of Plant and Soil Science

Texas Tech University

mailto:[email protected]

extraction and characterization of galactomannan from guar

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